A central theme of this proposal is based on the evolving concept that tight junction (TJ) proteins do not function simply as static, physical barrir forming elements but as signaling centers that regulate critical homeostatic functions within intestinal epithelial cells. This concept is consistent with findings in humans that have condition associated with dysregulated intestinal homeostasis such as ulcerative colitis and Crohn's Disease where altered TJ protein expression is associated with defective intestinal epithelial barrier function and extensive mucosal ulceration/wounding. The goal of this proposal is centered on elucidating fundamental mechanisms of how the tight junction protein JAM-A signals to control epithelial barrier and cell migration. It will specifically focus on identifyingnovel proximal signaling elements linked to JAM-A that regulate barrier during junctional assembly and during the steady state (Aim 1). It will also focus on elucidating how JAM-A regulates cell migration through effects on beta 1 integrin dynamics at the level of protein trafficking and small GTPase signaling. We have demonstrated that JAM-A mediated regulation of cell migration and barrier are dependent on dimerization of JAM-A in a cis configuration within the same cell. In aim 3 of this proposal, we will extend these important findings to determine the role of dimerization of JAM-A between cells in trans and if such interactions play a role in regulating cell migration and barrier. Knowledge gained from these studies will not only shed new light into mechanisms of outside-in signaling at the TJ that regulate permeability, migration and proliferation, but may also provide new ideas for therapeutic targets with diverse applications ranging from enhanced vaccine/drug delivery to wound healing/anti-inflammation or inhibition of cancer progression. PUBLIC HEALTH RELEVANCE: The goal of this proposal is centered on elucidating basic mechanisms of how a protein termed JAM-A that is abundantly expressed in the epithelial lining of the intestine controls leakiness of the barrier and how cells migrate to heal wounds. These studies have important implications in humans since disease conditions associated with altered expression of tight junction proteins including JAM-A, such as ulcerative colitis and Crohn's disease, are characterized by a leaky intestinal barrier and extensive mucosal injury.